1. Field of the Invention
The invention relates to an improved nucleic acid molecule encoding fungal immunomodulatory protein (FIP) that is better expressed in fungi, to vectors comprising the nucleic acid molecule, to hosts transformed with said vectors, to processes of expressing the protein of the invention in said transformed hosts, to the protein of the invention produced by said processes, to uses of said hosts comprising the protein of the invention and to a process of purifying FIP. The protein of the invention has wide immunomodulatory activity. Thus, the present invention further relates to uses of the protein of the invention in cosmetic or pharmaceutical compositions and to food or feed additives comprising the protein of the invention. Finally, the invention relates to the method of modulating immunological activities by orally administering FIP or proteins fused with FIP to a subject.
2. Description of the Prior Art
Lectins from Fungal fruit bodies or mycelia have immunomodulatory and hepatoprotective effects that have been suggested to be able to remove free radicals (Lin J. M. et al., Am J Chin Med. 1993; 21(1):59-69). (L)-galactoside-specific lectins from mistletoe enhance cytokine production in vitro and in vivo (Gabius H. J. et al., Anticancer Res. 1992 May-June; 12(3):669-75). In a test using Balb/C mouse as the disease model, lectins and recombinant lectins can inhibit tumor formation (Couraud P. O. et al., J. Biol. Chem. 1989; 264:1310-1316).
Ganoderma is a rare and valuable herb in Chinese medicine. It has been known in China for over 5,000 years as “Ling Zhi”. There are a variety of ganodenmas, including G. lucidum (red), G. applanatum (brown), G. tsugae (red), G. sinense (black), and G. oregonense (dark brown).
Several proteins from edible fungi such as Ganoderma Lucidium (Ling zhi or Reishi), Volvariella Volvacea (Chinese Mushroom), Flammulina Velutipes (Golden needle mushroom) share similar amino acid sequences and immunomodulatory functions. These proteins were named fungal immunomodulatory proteins (FIPs) (Ko J. L., Eur. J. Biochem. 1995; 228:224-249). Among all health foods in Chinese medicine, Ling Zhi is the best-studied fungus.
It has been known that Ling Zhi has anti-allergy (Chen H. Y et al., J. Med. Mycol. 1992; 33:505-512), hepatoprotective (Lin J. M. et al., Am J Chin Med. 1993; 21(1):59-69), anti-tumor effects (Wasser S P (1999), Crit. Rev Immunol 19:65-96) and immune advantages (Kino (1989), Journal of Biological Chemistry 264(1): 472-8). However, Ling Zhi is used restrictedly in the form of extract of raw material (Horner W. E. et al., Allergy 1993; 48:110-116) or small molecules (Kawagishi H., et al., Phytochemistry 1993; 32: 239-241).
One FIP purified from Ling Zhi, LZ-8 (Ling Zhi-8), has positive effects on systemic anaphylaxis (Kino K. et al., J. Biol. Chem. 1989; January 5; 264(1):472-8). LZ-8 has been used for the treatment of liver cancer and to prevent diabetes (Kino K. et al., J. Biol. Chem. 1989; January 5; 264(1):472-8). LZ-8 and another immunomodulatory protein FIP-five obtained from Flammulina Velutipes have similar amino acid sequences and folding structures similar to the heavy chain of immunoglobulin. Further, it has been shown that by enhancing the expression of LZ-8, these proteins show immunomodulatory activities and have positive effects on patients with systemic anaphylaxis (Ko J. L., Eur. J. Biochem. 1995; 228:224-249).
Baker's yeast is a safe microorganism widely used in traditional food processing (such as bread, wine). Baker's yeast is the typical model organism to study genetics, physiology and molecular biology. Because of its safety, baker's yeast was also successfully utilized to produce protein drugs such as Hirudin, Hemoglobulin, Urokinase, HSA (human serum albumin), IGF-I (insulin-like growth factor 1), GM-CSF (granulocyte macrophage colony stimulating factor), hepatitis B vaccine, etc. (Romanos M. A. et al., Yeast 1992; 8: 423-488). The proper host yeast used to produce the designate protein was selected according to the expression profile of the protein expressed. For example, Saccharomyces cerevisiae could be used in the production of food and protein drugs.
At the current stage, LZ-8 can only be obtained from Ling Zhi by a complicated and time-exhausting preparation protocol (such as extraction, U.S. Pat. No. 5,334,704). Although there is a great demand of Ling Zhi, natural Ling Zhi is rare and only grows on aged trees in steep mountains. Therefore, a technique to provide constant supply of high quality Ganoderma is needed. Currently there is no protocol for massive production of LZ-8 available.
Using the protocol described in the previous study (U.S. Pat. No. 5,334,704), the production of Ling Zhi immunomodulatory protein by extraction and purification is in lower efficiency and requires a higher cost. Other methods using E. coli to produce Ling Zhi immunomodulatory protein may have endotoxin contamination and the process of purification still requires a higher cost. Methods using mammalian cells to produce Ling Zhi immunomodulatory proteins require expensive medium and may meet viral or prion contamination.
U.S. Pat. No. 5,334,704 has disclosed that the glycoprotein isolated from ganoderma have immunosuppressive activity. The preparation of the glycoprotein includes steps of culturing ganoderma mycelia, extracting the resultant mycelia with an aqueous solvent and purifying the target protein from the resultant extract. The technique of the patent, which requires growing fungi and purifying the target protein from the fungi, requires a higher cost and is much more complicated than growing microorganism in vitro and is difficult to conduct industrial application.